Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

Surface properties of irreversibly sickled cells differ from those of the bulk of sickle cells Studies by partitioning in aqueous phase systems

Counter-current distribution (CCD) of red blood cells (RBC) from individuaks with homozygous sickle cell (HbSS) disease in a charge-sensitive aqueous dextran-poly(ethylene glycol) phase system, which fractionates cells on the basis of surface properties, indicates that the percentage of irreversibly sickled cells (ISC) increases and the percentage of reticulocytes decreases with increasing cell partition ratios. The high partition ratios of ISC correspond to those of older RBC when RBC from normal individuals are subjected to CCD. Our results thus indicate that ISC differ in surface properties from those of the bulk of sickle RBC (including reticulocytes) in the population and that the difference is, most likely, charge-related. While the question as to whether ISC are indeed old cells has not yet been unequivocally answered, this view finds support in the fact that the independent parameters of ISC surface properties, as reflected by partition ratios, and densities correlate as they do in older RBC from normal individuals.     

Activation enthalpy of diffusion for well fractionated dextrans in aqueous solutions

Interdiffusion coefficients have been determined for seven well defined dextran fractions in aqueous solutions at 20, 25, 30 and 35 degrees C. For dextrans with number average molecular weights (Mn) greater than about 20 000, a plot of the apparent activation energy of diffusion (ED) against Mn is given by: ED = 2.763 x 10(-5) Mn + 3.38. Similarly, for weigh molecular weight (Mw): ED = 1.889 x 10(-5) Mw + 3.61. The extrapolated ED values (3.38 and 3.61) are in reasonably good agreement with published data for the activation energy of self-diffusion for water.     

Characterization and antioxidant activity of an exopolysaccharide produced by Leuconostoc pseudomesenteroides JF17 from juçara fruits (Euterpe edulis Martius)

Juçara palm tree is considered an important species in the Atlantic forest ecosystem and the exploitation of its fruits is a main sustainable strategy adopted to its maintenance. Among several microorganisms in the juçara fruits, were found lactic acid bacteria (LAB) highly capable of synthesizing exopolysaccharides (EPS). Thus, the EPS synthesized by Leuconostoc pseudomesenteroides JF17 was characterized according to its chemical structure, physicochemical properties and antioxidant activity. HPLC analysis showed that the EPS consisted of a homopolysaccharide composed of glucose units, FT-IR spectroscopy revealed a dextran, confirmed by ¹H and ¹³C NMR spectra which showed the composition of the polysaccharide, d-glucose units bound by α-(1→6). The EPS showed high water retention capacity, 507.812 ± 47.471 %, and may be related to the porous structure revealed by SEM images. Thermal analysis indicated that the EPS had excellent thermal stability with degradation temperature of 320.8 °C and melting point at 279.81 °C, with the enthalpy of 106.557 J/g. The EPS also presented sequestration ability on different free radicals tested, indicating high antioxidant activity. The results suggest that L. pseudomesenteroides JF17 might be widely used in the production of dextran which has potential qualities for applications in the food industry.     

Dual character of Toll-like receptor signaling: Pro-tumorigenic effects and anti-tumor functions

As a major class of pattern-recognition receptors, Toll-like receptors (TLRs) play a critical role in defense against invading pathogens. Increasing evidence demonstrates that, in addition to infection, TLRs are involved in other important pathological processes, such as tumorigenesis. Activation of TLRs results in opposing outcomes, pro-tumorigenic effects and anti-tumor functions. TLR signaling can inhibit apoptosis and promote chronic inflammation-induced tumorigenesis. TLR activation in tumor cells and immune cells can induce production of cytokines, increase tumor cell proliferation and apoptosis resistance, promote invasion and metastasis, and inhibit immune cell activity resulting in tumor immune escape. In contrast, the engagement of other TLRs directly induces growth inhibition and apoptosis of tumor cells and triggers activation of immune cells enhancing anti-tumor immune responses. Thus, the interpretation of the precise function of each TLR in tumors is very important for targeting TLRs and using TLR agonists in tumor therapy. We review the role of TLR signaling in tumors and discuss the factors that affect outcomes of TLR activation.     

Interaction between dextran and human low density lipoproteins (LDL) observed using laser light scattering

Dextran infusions in humans lead to a reduction of low density lipoproteins (LDL) in the plasma compartment. The interaction of dextran with human LDL was investigated in vitro by static and dynamic light scattering. The experiments were performed with human LDL (apoB concentration 0.75 g l⁻¹) and dextran (M_w=40 000 and 70 000 g mol⁻¹) at 25°C. The dextran concentrations after mixing were 10 and 50 g l⁻¹. The hydrodynamic radius for native LDL was found to be R_H=12.9 nm. The addition of dextran induces the formation of LDL associates with a mean radius of R_H≈200 nm. These findings show that even non-sulphated polysaccharides interact with LDL. The dextran-dependent formation of LDL associates detected in vitro could be the reason for the in vivo effect of dextran on the lipid metabolism.     

Histone deacetylase complexes: functional entities or molecular reservoirs

Using the dextran-binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD-tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross-linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water-soluble dextran. The purity of the eluted proteins after this one-step affinity purification was 90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins.     

In vitro measurement of nuclear permeability changes in apoptosis

In the eukaryotic cell, exchange of biomolecules between nucleus and cytoplasm is a highly regulated process which responds sensitively to changes of the environment. One well-known cellular response to environmental challenges is cell death by apoptosis. In fact, apoptosis has been shown to affect the nucleocytoplasmic transport machinery, in particular the nuclear pore, by modulating its size exclusion limit for passive diffusion. The underlying molecular factors are still unknown, mainly because of the lack of a suitable system to detect and quantitate the apoptotic effects on the nuclear pore. Here we present an assay that was designed to measure alterations of the permeability of the nuclear envelope under apoptotic conditions. The assay is based on the well-established technique of selective permeabilization of the plasma membrane with digitonin and allows assessment of permeability changes in nonfixed samples. It comprises a computer program, called Nuclear Permeability Assay, for the quantitation of the nuclear fluorescence signal, which may be generally employed for the evaluation of in vitro transport systems using semipermeabilized cells, such as assays for nuclear import and export.     

Dextran–estrone conjugate: synthesis and in vitro release study

A novel hormone conjugate has been prepared by a coupling reaction between modified estrone and dextran. In order to provide a suitable reactive estrone derivative for coupling with dextran and a spacer between the drug carrier and the hormone, the steroidal sex hormone was succinylated by reaction with succinic anhydride. Subsequently, the carboxylic acid terminal of succinylated estrone was further reacted with thionyl chloride to replace the hydroxy group with chlorine to make a better leaving group. The ester bond was employed as labile linkage between the hormone and the biopolymer carrier backbone so that the coupled estrogen could be released from the conjugate via ester hydrolysis. Structures of the modified estrone and dextran–estrone conjugate were determined by elemental analysis and by FTIR, ¹H and ¹³C NMR spectroscopies. The degree of substitution (D.S.) per anhydroglucose (AHG) unit was 0.33 (11.0 mol-% of estrone moieties), as calculated from the ¹H NMR spectrum. In vitro hydrolysis of the conjugate in aqueous phosphate buffer/ethanol solutions at pH 8.0 and 7.4 and 37°C released estrone was completed within a few days and followed zero-order kinetics.     

pH-imprinted lipase catalyzed synthesis of dextran fatty acid ester

The application of enzymatic catalysis for the synthesis of polysaccharide-based surfactants was investigated. The polysaccharide dextran, a neutral bacterial polysaccharide consisting of -1,6 linked glucose units, was chemically modified by the attachment of hydrophobic groups through a transesterification reaction with a vinyl decanoate. A screening of commercially available lipases and protease for the synthesis of amphiphilic polysaccharides in DMSO suggested that lipase AY from Candida rugosa modified dextran T-40 with vinyl decanoate at the highest conversion. A pH-adjustment in a phosphate buffer at pH 7.5 prior to use is crucial to make this enzyme active in DMSO. The effect of enzyme concentration and mole ratio of fatty ester to dextran T-40 on the conversion and the rate of reaction were studied. Finally, investigation of the kinetics and regioselectivity of lipase AY-catalyzed modification offer a possibility to regulate the position and the extent of hydrophobic group attached to dextran. These two properties are fundamental for controlling the physico-chemical properties of the final polymeric surfactants.